Increase in plasma thrombin-activatable fibrinolysis inhibitor may not contribute to thrombotic tendency in antiphospholipid syndrome because of inhibitory potential of antiphospholipid antibodies toward TAFI activation.

The causes of thrombosis in antiphospholipid syndrome (APS) remain unknown, though several hypotheses in regard to hypofibrinolysis have been proposed. To clarify the mechanism, we measured plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in APS patients. Both the TAFI antigen (TAFI:Ag) level measured with an ELISA, and thrombin-thrombomodulin-dependent TAFI activity (TAFI:Ac) were elevated in 68 APS patients as compared with those in 66 healthy controls, though they were lower than those in 46 patients with autoimmune diseases. As for the influence of antiphospholipid antibodies (aPL) on TAFI levels, the mean TAFI:Ac level in 39 SLE patients positive for APS was significantly lower than that in 27 SLE patients without APS, whereas there was no difference in TAFI:Ag between those groups. Furthermore, purified IgG from patients positive for aPL, and monoclonal aPL (EY2C9 and 23-1D) inhibited the activation of TAFI in a concentration dependent manner. These results suggest that aPL inhibits TAFI activation by affecting the function of thrombomodulin-thrombin complex through phospholipids. Although TAFI in plasma is elevated in autoimmune diseases including APS, we concluded that an elevated level is not likely a risk factor for thrombosis in APS patients, because of the inhibition of TAFI activation by aPL.

Plasma soluble fibrin monomer complex as a marker of coronary thrombotic events in patients with acute myocardial infarction.

Soluble fibrin monomer appears in the bloodstream during the extremely early stage of blood coagulation and generally forms a complex with fibrinogen, termed soluble fibrin monomer complex (FMC). Determination of FMC can provide information regarding the state of thrombotic diseases; thus it is important to investigate whether FMC serves as an early indicator of myocardial infarction (MI). We investigated hemostatic and fibrinolytic parameters including FMC to determine their capabilities for indicating thrombotic conditions in the coronary artery. Blood samples from 47 patients with acute MI were obtained within 48 hours (acute phase) and during 120 - 600 hours (recovery phase), respectively, after MI onset. Plasma FMC was significantly elevated in the acute phase, compared with that during the recovery phase and in healthy controls (p = 0.001), suggesting that its elevation indicates thrombotic events in the coronary artery of MI patients. D-dimer, a marker of thrombus formation accompanied with fibrinolysis, was increased in both phases in the patients. In addition, FMC and D-dimer were significantly increased within 24 hours after onset as compared to 24 - 48 hours (p = 0.003 and p = 0.011). Furthermore, cardiac troponin T, a marker of myocardial damage, was significantly higher after 24 hours than within the first 24 hours (p = 0.001). Receiver operating characteristic (ROC) analysis of FMC for early MI diagnosis indicates that FMC, rather than D-dimer, is a better marker within 24 hours of onset. Measuring plasma FMC may be useful for early diagnosis of MI recurrence and deciding primary treatment.

New therapeutic option for thromboembolism--dabigatran etexilate.

BACKGROUND: Thrombin plays a key role in blood coagulation and haemostasis; thus its inhibition has been identified as a reasonable target to block the coagulation cascade. Direct thrombin inhibitors are potential prophylactic agents for venous thromboembolism and arterial thrombosis, which often accompany operative procedures and cardiac disease, especially orthopedic surgery and atrial fibrillation, respectively. New orally available anticoagulant agents with a wide therapeutic window are keenly anticipated because warfarin and heparins have some disadvantages, and recent progress in pharmaceutical techniques has led to the development of orally administered direct thrombin inhibitors. OBJECTIVES: In this review, we discuss the usefulness of dabigatran etexilate as a new therapeutic option for preventing thromboembolism, including chemistry, pharmacokinetics, and pharmacodynamics, from the results of recent clinical studies. METHODS: We systematically focused on relevant published studies, as data from recent clinical studies were difficult to obtain owing to their ongoing status. CONCLUSIONS: Dabigatran etexilate is a promising new oral anticoagulant that offers greatly expanded therapeutic options for both patients and physicians.

[Study on usefulness of different APTT test kit in variable coagulopathy].

A number of test kits are available for measuring activated partial thromboplastin time (APTT) and are used to screen for intrinsic coagulation reactions. However, results obtained with the same sample by different test kits often vary, causing confusion regarding potential hemostatic activity in the specimen. We investigated the usefulness of 6 different APPT kits, which utilize various phospholipids and activators, to detect prolonged clotting time in plasma from subjects with abnormal coagulopathy, including lupus anticoagulant(LA). In samples from subjects with intrinsic coagulation factor deficiencies and subjects taken heparin, the abnormal APTT detection ratio was high regardless of the kit used, thus any would be acceptable for measuring APTT in such patients. In contrast, that ratio in patients with von Willebrand disease was relatively low regardless of the kit, probably because factor VIII activities in those patients were slightly decreased. The ratio of detected subjects with LA and subjects taking warfarin varied among the APTT kits, however, those that utilized synthetic phospholipids were useful for the detection of LA. Our results suggest that an APTT kit should be selected according to the kind of disorder in the patient. Further, kits that employ synthetic phospholipids are useful for detecting abnormal coagulopathy in patients with intrinsic coagulation factor deficiencies and patients taken heparin, as well as for detection of LA.

Soluble fibrin monomer degradation products as a potentially useful marker for hypercoagulable states with accelerated fibrinolysis.

BACKGROUND: Fibrin monomer (FM) and its complex (sFC) exist at high concentrations in hypercoagulable state blood. Two novel immunoassays for sFC (SF and FMC) using specific monoclonal antibodies (IF-43 and F405) were recently developed. METHODS: We measured the concentrations of thrombotic markers in 103 patients with DIC and thrombotic disorders. RESULTS: We found that the concentration of FMC was approximately 3.35-fold greater than that of SF. In patients with a high FMC/SF ratio, FDP and D dimer concentrations were increased, suggesting that the discrepancy in sFC concentrations was caused by fibrinolytic activity. Further, plasma samples from those patients were found to contain the X- and Y-fragments of FM in addition to FM and sFC in a Western blotting assay using F405, which binds with those fragments. In an in vitro study, FM formed from pooled plasma containing EDTA was degraded to the X- and Y-fragments of FM by fibrinolytic activity, and we termed those FM degradation products (FMDP). CONCLUSION: Determination of FMDP is important for diagnosis of thrombogenic conditions associated with fibrinolysis, such as in patients with DIC, and it may serve as a useful marker for hypercoagulable states with accelerated fibrinolysis.

Molecular dynamics calculations of wild type vs. mutant protein C: relationship between binding affinity to endothelial cell protein C receptor and hereditary disease.

Molecular dynamics simulations of the protein C gamma-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 --> Lys) in the protein C Gla domain. Our results suggest that the Gla 25 --> Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca2+ ion.

One novel and one recurrent mutation in the PROS1 gene cause type I protein S deficiency in patients with pulmonary embolism associated with deep vein thrombosis.

We investigated the molecular basis of type I protein S (PS) deficiency in two unrelated Japanese families, in which both probands developed pulmonary embolism associated with deep vein thrombosis. Nucleotide sequencing of amplified DNA revealed distinct point mutations in the PROS1 gene of the probands, which were designated protein S Sapporo 1 and protein S Sapporo 2. Additional mutations in the PROS1 gene were excluded by DNA sequencing of all exons and intron/exon boundaries. In the 25-year-old Japanese male patient who carried protein S Sapporo 1, we identified a heterozygous A-to-T change in the invariant ag dinucleotide of the acceptor splice site of intron f of the PROS1 gene. This mutation is a novel splice site mutation that impairs normal mRNA splicing, leading to exon 7 skipping, which was confirmed by platelet mRNA analysis. Translation of this mutant transcript would result in a truncated protein that lacks the entire epidermal growth factor-like domain 3 of the PS molecule. In a 31-year-old Japanese male and his younger brother who each carried protein S Sapporo 2, we detected a previously described heterozygous T-to-C transition at nucleotide position 1147 in exon 10 of the PROS1 gene, which predicts an amino acid substitution of tryptophan by arginine at residue 342 in the laminin G1 domain of the PS molecule. Both mutations would cause misfolding of the PS protein, resulting in the impairment of secretion, which is consistent with the type I PS deficiency phenotype.

[Phosphatidylserine-dependent anti-prothrombin antibody as a new marker for the diagnosis of antiphospholipid syndrome].

Antiphospholipid syndrome (APS) is an autoimmune disorder in which vascular thrombosis and recurrent pregnancy loss occur in patients with antiphospholipid antibodies(aPL). Measurements of the beta2-glycoprotein I-dependent anticardiolipin antibody(aCL) and lupus anticoagulant(LAC) are the only laboratory tests available for the diagnosis of APS. Recently, phosphatidylserine-dependent antiprothrombin antibody(aPS/PT) has been detected. aPS/PT was measured by ELISA using the phosphatidylserine-prothrombin complex as an antigen immobilized on ELISA plates in the presence of CaCl2. In our study of 219 patients with APS and autoimmune diseases, the prevalence of aPS/PT-IgG in those with APS was 42.2%, which was significantly higher than that(4.6%) in patients with autoimmune diseases. Furthermore, aPS/PT was closely associated with APS manifestations with an odds ratio (OR) of 2.92 (95% confidence interval (95% CI): 1.33 to approximately 6.40), whereas the OR for aCL was 2.06 (95% CI: 0.91 to approximately 4.66). In addition, aPS/PT-IgG was strongly correlated with the presence of LAC as detected with a diluted Russell viper venom time test (dRVVT) (OR: 38.2, 95% CI: 13.4 to approximately 109.1). The monoclonal antibody (23-1D) of aPS/PT also prolonged the clotting time in LAC tests (aPTT, dRVVT, and kaolin clotting time) in a concentration-dependent manner. In conclusion, aPS/PT is more closely associated with manifestations of APS and LAC, and positive results from an aPS/PT test can mark thrombotic events in APS patients. The determination of aPS/PT in clinical practice, in conjunction with that of other aPL, may improve the likelihood of recognizing APS.

Factor Xa inhibitors: new anti-thrombotic agents and their characteristics.

Factor Xa (FXa) is a key enzyme that is positioned at the convergence of the intrinsic and extrinsic pathways in the blood coagulation cascade, and inactivation by a specific FXa inhibitor effectively prevents the generation of thrombin. Various types of low molecular weight (LMW) heparin, which function as semi-selective and indirect FXa inhibitors, are replacing unfractionated heparin (UFH) as agents for the prevention and treatment of venous thromboembolism (VTE), as well as in initial treatment for coronary events. Of those, heparinoid has been shown to be safer and more effective for the prevention of postoperative VTE than UFH, especially for treatment of heparin-induced thrombocytopenia (HIT). Further, synthetic pentasaccharide has been found to offer advantages over current thromboprophylactic regimens in a number of patients undergoing major orthopedic surgery. Other studies have shown that pentasaccharide is more effective for overall VTE in comparison with LMW heparin, though it was also associated with an increased rate of major bleeding. Synthetic, selective, and direct inhibitors to FXa, such as DX-9065a, are highly potent and orally bioavailable antithrombotic agents that have demonstrated an improved side effect profile, probably by allowing sufficient thrombin to remain for platelet activation and normal hemostasis, while preventing pathological thrombus formation. For thrombosis therapy, the most desirable type of antithrombotic agent is an orally active drug that has a broad range of effective doses and no hemorrhagic side effects. Presently, many types of direct inhibitors are in various stages of clinical trials and expected to provide significant benefits as compared to currently utilized therapy strategies.

Protein C Sapporo (protein C Glu 25 --> Lys): a heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor.

Interaction of the gamma-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G to A transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25-->Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25-->Lys mutation induces type IIb protein C deficiency in individuals.